JP7101982B2 - Pharmaceutical composition for the treatment of cerebral infarction - Google Patents

Description

The present application relates to pharmaceutical compositions for the treatment of cerebral infarction.

Cerebral infarction is generally a disease characterized by cerebral ischemia caused by obstruction or stenosis of cerebral arteries, resulting in necrosis or near necrosis of brain tissue due to lack of oxygen or nutrients. Cerebral infarction includes "lacuna infarction" caused by small arterial lesions in the brain, "atherothrogenic cerebral infarction" caused by atherosclerosis of relatively large arteries in the neck or skull, and "cardiogenic brain" caused by heart disease. Disease types such as "embolism" are included. Cerebral infarction is a disease with a high mortality rate, and once it develops, it is likely to leave sequelae, which poses a major problem in terms of patient QOL, welfare, and medical economy.

For acute cerebral infarction, thrombolytic treatment with tissue plasminogen activator (tPA or t-PA) is the first choice. Plasminogen is an enzyme that strongly dissolves blood clots formed in blood vessels. Treatment is performed to dissolve the thrombus and restore the blood flow by administering tPA after the cerebral infarction caused by the thrombus formed in the cerebral artery (thrombolytic therapy). It has been reported that 40-50% of patients recover to the point where they can live independently by tPA treatment. However, if the time from the onset to the administration of tPA is long, it may cause cerebral hemorrhage and can be used only for patients within 4.5 hours after the onset. In addition, recently, endovascular treatment for removing thrombus by mechanical thrombus recovery therapy is being carried out for treatable patients within 8 hours from the onset. In any case, further treatment improvement rate is strongly desired from clinical practice, and it is extremely important to develop a new treatment method.

Certain 4-amino-naphthalene-1-sulfonic acid derivatives have VCP (valosin-containing protein) ATPase inhibitory activity and are expected to have therapeutic effects on various diseases (Patent Document 1). In particular, it is known to be effective in treating or preventing eye diseases and leptin resistance (Patent Documents 2 to 5).

International Publication No. 2012/014994 Pamphlet International Publication No. 2012/043891 Pamphlet International Publication No. 2014/12495 Pamphlet International Publication No. 2015/129809 Pamphlet International Publication No. 2015/033981 Pamphlet

An object of the present application is to provide a pharmaceutical composition for the treatment of cerebral infarction.

The present inventors have found that a VCP regulator has a protective effect on cerebral cortical neurons and is effective in treating cerebral infarction.

〔式中、
Ｒａはハロ、ヒドロキシ、アルキル、ハロ置換アルキル、アリール、ハロまたはアルキル置換アリール、アルコキシ、ヒドロキシまたはカルボキシ置換アルコキシ、アリールオキシ、ハロまたはアルキル置換アリールオキシ、ＣＨＯ、Ｃ（Ｏ）－アルキル、Ｃ（Ｏ）－アリール、Ｃ（Ｏ）－アルキル－カルボキシル、Ｃ（Ｏ）－アルキレン－カルボキシエステルおよびシアノから成る群から選択され、
ｍは０～４から選択される整数である〕
の化合物またはそのエステル、オキシド、薬学的に許容される塩もしくは溶媒和物を含む、脳梗塞の処置用の医薬組成物を提供する。 Therefore, in some embodiments, the present application describes the formula (I) :.

[During the ceremony,
Ra is halo, hydroxy, alkyl, halo-substituted alkyl, aryl, halo or alkyl-substituted aryl, alkoxy, hydroxy or carboxy-substituted alkoxy, aryloxy, halo or alkyl-substituted aryloxy, CHO, C (O) -alkyl, C (O). ) -Aryl, C (O) -alkyl-carboxyl, C (O) -alkylene-carboxyester and selected from the group consisting of cyano
m is an integer selected from 0 to 4]
To provide a pharmaceutical composition for the treatment of cerebral infarction, which comprises the compound of the above or an ester, oxide, pharmaceutically acceptable salt or solvate thereof.

The present disclosure provides pharmaceutical compositions for the treatment of cerebral infarction.

The procedure of an in vitro experiment using primary cerebral cortex neurons is shown. It shows the viability of neurons after oxygen glucose deficiency (OGD) treatment in the presence and absence of KUS121. Shows the MAP2-positive areas in the presence and absence of KUS121 in neurons after OGD treatment. The ATP levels in the presence and absence of KUS121 in neurons after OGD treatment are shown. The expression level of C / EBP homologous protein (CHOP) in the presence and absence of KUS121 in the neurons treated with tunicamycin is shown. The procedure of an in vivo experiment using mice is shown. The time spent on the rotarod of C57BL / 6 mice treated with KUS121 or vehicle after inducing transient localized cerebral ischemia is shown. The infarct volume in the brain of C57BL / 6 mice treated with KUS121 or a vehicle after inducing transient localized cerebral ischemia is shown. The infarct volume in the brain of CB-17 mice induced transient localized cerebral ischemia after administration of KUS121 or vehicle is shown. It is the result of Western blotting showing the expression level of NeuN in the cerebral cortex of CB-17 mice that induced transient localized cerebral ischemia after administration of KUS121 or a vehicle.

In the present disclosure, when a number is accompanied by the term "about", it is intended to include a range of ± 10% of that value. For example, "about 20" is assumed to include "18-22". The range of numbers includes all numbers between the end points and the numbers at the end points. The "about" for a range applies to both ends of the range. Therefore, for example, "about 20 to 30" includes "18 to 33".

Unless otherwise specified, the terms used in this disclosure have meanings generally understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology and the like. The following are definitions of some terms used in this disclosure, but these definitions supersede the general understanding in this disclosure.

"Alkyl" means a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. Alkyl means, for example, linear and branched hydrocarbyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and neopentyl. Not limited.

The prefix "substitution" of a group means that one or more hydrogen atoms of the group are substituted with the same or different designated substituents.

"Alkylene" means a divalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. The alkylene group contains a branched chain and a linear hydrocarbyl group.

"Alkoxy" means the group of -O-alkyl, where alkyl is defined in the present disclosure. Alkoxy means, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy and n-pentoxy, but is not limited thereto.

"Aryl" means a monovalent aromatic carbocyclic group of 6 to 14 carbon atoms having one ring (eg, phenyl) or multiple fused rings (eg, naphthyl or anthryl). Aryl groups typically include phenyl and naphthyl.
"Aryloxy" means the group of -O-aryl, where aryl is defined herein, including, for example, phenoxy and naphthoxy.

"Cyanide" means the group of -CN.
"Carboxylic" or "carboxylic" means -COOH or a salt thereof.
"Carboxyester" means the group of -C (O) O-alkyl, where alkyl is defined in the present disclosure.
"Halo" means halogen, in particular fluoro, chloro, bromo and iodine.
"Hydroxy" means the group of -OH.

Unless otherwise specified, the nomenclature of substituents not explicitly defined in the present disclosure is to name the terminal portion of the functional group and then the functional group adjacent to the binding point. For example, the substituent "arylalkyloxycarbonyl" means (aryl)-(alkyl) -OC (O)-.

The compound of formula (I) may be enantiomeric or diastereomeric, depending on the substitution pattern. The compound of formula (I) may be racemic or may be separated into pure stereoisomerically by a known method. Certain compounds can be tautomers.

"Ester" means an ester that can be hydrolyzed in vivo, including those that are easily degraded by the human body to release the parent compound or salts thereof. Suitable ester groups are, for example, derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic acid, alkenoic acid, cycloalkanoic acid and alkanedioic acid (where each alkyl or alkenyl group is, for example, 6 (Has no more than one carbon atom). Specific examples of esters include formic acid esters, acetic acid esters, propionic acid esters, butyric acid esters, acrylic acid esters and ethyl succinic acid esters.
"Oxide" means that the nitrogen ring atom of the heteroaryl group is oxidized to form N-oxide.

A "pharmaceutically acceptable salt" can be a salt of a compound of formula (I) with an inorganic or organic acid. Preferred salts are salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or organic carboxylic acids or sulfonic acids such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, It is a salt with malic acid, citric acid, tartrate acid, lactic acid, benzoic acid or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid or naphthalenedisulfonic acid.

Also, pharmaceutically acceptable salts are salts with conventional bases, such as alkali metal salts (eg sodium or potassium salts), alkaline earth metal salts (eg calcium or magnesium salts), or ammonia or organic amines (eg calcium or magnesium salts). For example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholin, dihydroabiethylamine, methylpiperidin, L-arginine, creatine, choline, L-lysine, ethylenediamine, benzatin, ethanolamine, meglumin or tromethamine. ) Can be an ammonium salt, especially a sodium salt.

The "solvate" means a compound of formula (I) that forms a complex by coordination with a solvent molecule in a solid or liquid state. A suitable solvate is a hydrate.

References to "compounds of formula (I)" in the present disclosure are intended to include esters, oxides, pharmaceutically acceptable salts and solvates thereof, unless inappropriate in the context.

In certain embodiments, in formula (I), Ra is independently selected from the group consisting of halo, hydroxy, alkyl, halo-substituted alkyl and alkoxy.
In certain embodiments, in formula (I), Ra is independently selected from the group consisting of halos and alkyls, respectively.
In one embodiment, in formula (I), there are two Ras, one is halo and the other is alkyl.

In certain embodiments, the compounds of formula (I) are selected from the compounds in Table 1 below:

で表される４－アミノ－３－［６－（４－フルオロ－２－メチルフェニル）ピリジン－３－イルアゾ］ナフタレン－１－スルホン酸、または、そのエステル、オキシド、薬学的に許容される塩もしくは溶媒和物であり、特にナトリウム塩が好ましい。 In certain embodiments, the active ingredient of the pharmaceutical composition is formulated by:

4-Amino-3- [6- (4-fluoro-2-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid represented by, or an ester, oxide, or pharmaceutically acceptable salt thereof. Alternatively, it is a solvate, and a sodium salt is particularly preferable.

The properties of the compound of formula (I), in particular the above-mentioned compounds and methods of synthesis, are described in detail in International Publication No. 2012/014994 Pamphlet (Patent Document 1).

In the present disclosure, "cerebral infarction" means brain damage due to cerebral ischemia, including, but not limited to, lacunar infarction, atherothrombotic cerebral infarction and cardiogenic cerebral embolism. Causes of cerebral ischemia include, but are not limited to, cerebral thrombosis, cerebral embolism, vasospasm, hypotension, hypoxemia, and the like. In the present disclosure, symptoms related to cerebral infarction such as neuropathy, higher dysfunction, and emotional disorder or aftereffects of cerebral infarction are also included in the term of cerebral infarction.

In the present disclosure, "treatment" includes any medical intervention aimed at curing, remission, alleviating, ameliorating and / or temporary remission of a disease or condition. For example, "treatment" includes medical interventions for a variety of purposes, including delaying or stopping the progression of a disease, regressing or eliminating lesions, or preventing recurrence.

Targets for treating the disease include animals, typically mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.), especially humans.

The administration method of the pharmaceutical composition is not particularly limited, but it can be administered through general administration routes such as oral administration, non-enteral administration, injection, and infusion, and the composition can be in a dosage form suitable for each administration route. .. Intrathecal and epidural administration, or intraventricular administration is also possible. In certain embodiments, the pharmaceutical composition is administered intravenously.

Dosage forms for oral administration include granules, fine granules, powders, coated tablets, tablets, suppositories, powders, capsules, microcapsules, chewables, liquids, suspensions, emulsions and the like. .. As the dosage form for administration by injection, a general dosage form of a pharmaceutical preparation such as an intravenous injection, an infusion administration, and a preparation that prolongs the release of an active substance can be adopted. Dosage forms for intravenous or drip infusion include aqueous and non-aqueous injection solutions (which may include antioxidants, buffers, bacteriostatic agents, isotonic agents, etc.); and aqueous and non-aqueous injections. Examples include suspensions (which may include suspending agents, thickeners, etc.). The dosage form for injection may be provided in a closed ampoule or vial, may be provided as a lyophilized product, and may be prepared by adding a sterile liquid (eg, water for injection) immediately before use. Injection solutions or suspensions may be prepared from powders, granules or tablets.

These dosage forms are produced by formulating them by a conventional method. Further, various pharmaceutically acceptable preparation substances can be blended as required in the preparation. The substance for preparation can be appropriately selected depending on the dosage form of the preparation, and for example, a buffering agent, a surfactant, a stabilizer, a preservative, an excipient, a diluent, an additive, a disintegrant, a binder, etc. Examples thereof include coating agents, lubricants, lubricants, flavoring agents, sweeteners, solubilizers and the like.

The dose and frequency of administration of the pharmaceutical composition depend on the animal species, health condition, age, body weight, administration route, administration form, etc. of the administration target so that the effective amount of the compound of the formula (I) is administered to the subject. Can be set appropriately by those skilled in the art. The effective amount in certain situations can be easily determined by routine experimentation and is within the skill and judgment of the usual clinician. For example, a compound of formula (I) of about 0.001 to about 1000 mg / kg body weight / day, about 0.01 to about 300 mg / kg body weight / day, or about 0.1 to about 100 mg / kg body weight / day is administered. Can be. For example, the pharmaceutical composition may be administered once, multiple times, or continuously.

In certain embodiments, the pharmaceutical composition is administered during the acute phase of cerebral infarction. "Acute phase" means the period within 14 days of the onset of cerebral infarction. The pharmaceutical compositions of the present disclosure are such that after a subject develops a cerebral infarction, for example, promptly after the subject develops a cerebral infarction, for example, within about 3 hours, within about 4.5 hours, within about 6 hours from the onset. , Can be administered within about 8 hours, within about 12 hours or within about 24 hours. In the present disclosure, it is intended that the time at which the cause of cerebral ischemia (eg, occlusion or stenosis of a cerebral artery) occurs is regarded as the time of onset of cerebral infarction. In certain embodiments, the pharmaceutical composition is administered single or multiple times during the acute phase of cerebral infarction. In certain embodiments, the pharmaceutical composition is administered continuously over the entire or part of the acute phase of cerebral infarction. In certain embodiments, the pharmaceutical composition is administered before inflammation due to cerebral infarction occurs. The presence or absence of inflammation can be determined by, for example, an inflammatory marker such as blood CRP or IL-1β.

In certain embodiments, the pharmaceutical composition may be administered immediately before or after reperfusion treatment of the occluded blood vessel (eg, by administration of a thrombolytic agent or surgery). "Immediately before reperfusion therapy" means minutes to tens of minutes before reperfusion therapy, for example, about 60 minutes, about 30 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5. It means the time point from 1 minute or about 3 minutes before the reperfusion treatment. "Immediately after the reperfusion treatment" means several minutes to several tens of minutes after the reperfusion treatment, for example, about 60 minutes, about 30 minutes, about 20 minutes, about 15 minutes, and about 10 minutes. It means the time point after 1 minute, about 5 minutes, or about 3 minutes. In certain embodiments, the pharmaceutical composition is administered at the same time as the reperfusion procedure. Alternatively, reperfusion therapy may be performed while the pharmaceutical composition is continuously administered.

In certain embodiments, once in the acute phase, about 1 to about 1000 mg / kg body weight, about 5 to about 500 mg / kg body weight, about 10 to about 300 mg / kg body weight, about 25 to about 200 mg / kg body weight, for example about 100 mg. The compound of formula (I) of / kg body weight can be administered intravenously.

The compounds of formula (I) can be used alone or in combination with one or more additional active ingredients, in particular the active ingredient for the treatment of cerebral infarction. Active ingredients suitable for concomitant use include, for example, thrombolytic agents (eg, tPA, rtPA (eg, alteplase, moneprase, pamiteplase, desmoteplase, reteplase, tenecteprase), urokinase), antiplatelet agents (eg, ozagrel sodium, aspirin, clopidogrel, etc.) Syrostazole), selective thrombin inhibitors (eg algatroban), heparin, low molecular weight heparin, heparinoids, brain protectants (eg edarabon), hypertonic glycerol, hypertonic mannitol, vasodilators, anti-inflammatory agents, statins, etc. In particular, rtPA, in particular alteplase, is included, but is not limited to these.

"Combination" of ingredients is used not only for the use of dosage forms containing all ingredients and the use of combinations of dosage forms containing each ingredient separately, but also for the treatment of cerebral infarction. As long as it means that each component is administered at the same time or one of the components is delayed. Two or more additional active ingredients can also be used in combination.

In addition to the administration of the compound of formula (I), treatments other than drug therapy can also be performed. Suitable treatments include, for example, mechanical thrombosis recovery therapy, extracranial decompression therapy, emergency carotid intimal ablation, acute recanalization therapy, acute cervical carotid blood circulation reconstruction (angioplasty / stent placement). ), Etc., genetic therapy, regenerative medicine, etc. are included.

In some embodiments, a method of treating a stroke is provided that comprises administering a compound of formula (I) to a subject in need of treatment of the stroke.
In some embodiments, compounds of formula (I) are provided for use in the treatment of cerebral infarction.
In some embodiments, the use of a compound of formula (I) for treating a cerebral infarction is provided.
In some embodiments, the use of a compound of formula (I) in the manufacture of a pharmaceutical composition for the treatment of cerebral infarction is provided.

〔式中、
Ｒａはハロ、ヒドロキシ、アルキル、ハロ置換アルキル、アリール、ハロまたはアルキル置換アリール、アルコキシ、ヒドロキシまたはカルボキシ置換アルコキシ、アリールオキシ、ハロまたはアルキル置換アリールオキシ、ＣＨＯ、Ｃ（Ｏ）－アルキル、Ｃ（Ｏ）－アリール、Ｃ（Ｏ）－アルキル－カルボキシル、Ｃ（Ｏ）－アルキレン－カルボキシエステルおよびシアノから成る群から選択され、
ｍは０～４から選択される整数である〕
の化合物またはそのエステル、オキシド、薬学的に許容される塩もしくは溶媒和物を含む、脳梗塞の処置用の医薬組成物。
［２］Ｒａが、それぞれ独立して、ハロ、ヒドロキシ、アルキル、ハロ置換アルキルおよびアルコキシから成る群から選択される、第１項に記載の医薬組成物。
［３］Ｒａが、それぞれ独立して、ハロおよびアルキルから成る群から選択される、第１項または第２項に記載の医薬組成物。
［４］Ｒａが２個存在し、一方がハロであり、他方がアルキルである、第１項～第３項のいずれかに記載の医薬組成物。
［５］式（Ｉ）の化合物が表１に記載の化合物から選択される、第１項～第４項のいずれかに記載の医薬組成物。
［６］式（Ｉ）の化合物が、４－アミノ－３－［６－（４－フルオロ－２－メチルフェニル）ピリジン－３－イルアゾ］ナフタレン－１－スルホン酸である、第１項～第５項のいずれかに記載の医薬組成物。
［７］４－アミノ－３－［６－（４－フルオロ－２－メチルフェニル）ピリジン－３－イルアゾ］ナフタレン－１－スルホン酸ナトリウム塩を含む、第１項～第６項のいずれかに記載の医薬組成物。
［８］脳梗塞が、ラクナ梗塞、アテローム血栓性脳梗塞または心原性脳塞栓症である、第１項～第７項のいずれかに記載の医薬組成物。
［９］脳梗塞がラクナ梗塞である、第１項～第８項のいずれかに記載の医薬組成物。
［１０］脳梗塞の急性期に投与される、第１項～第９項のいずれかに記載の医薬組成物。
［１１］脳梗塞後の再灌流処置の直前または直後に投与される、第１項～第１０項のいずれかに記載の医薬組成物。
［１２］脳梗塞後の再灌流処置と同時に投与される、第１項～第１０項のいずれかに記載の医薬組成物。
［１３］再灌流処置が血栓溶解剤の投与である、第１１項または第１２項に記載の医薬組成物。
［１４］再灌流処置が外科手術である、第１１項または第１２項に記載の医薬組成物。
［１５］単回投与される、第１項～第１４項のいずれかに記載の医薬組成物。
［１６］複数回投与される、第１項～第１４項のいずれかに記載の医薬組成物。
［１７］持続投与される、第１項～第１４項のいずれかに記載の医薬組成物。
［１８］脳梗塞の発症から約２４時間以内に投与される、第１項～第１７項のいずれかに記載の医薬組成物。
［１９］脳梗塞の発症から約１２時間以内に投与される、第１項～第１８項のいずれかに記載の医薬組成物。
［２０］脳梗塞の発症から約８時間以内に投与される、第１項～第１９項のいずれかに記載の医薬組成物。
［２１］脳梗塞の発症から約６時間以内に投与される、第１項～第２０項のいずれかに記載の医薬組成物。
［２２］脳梗塞の発症から約４.５時間以内に投与される、第１項～第２１項のいずれかに記載の医薬組成物。
［２３］脳梗塞の発症から約３時間以内に投与される、第１項～第２２項のいずれかに記載の医薬組成物。
［２４］約１～約１０００ｍｇ／ｋｇ体重の式（Ｉ）の化合物を投与するための、第１項～第２３項のいずれかに記載の医薬組成物。
［２５］約５～約５００ｍｇ／ｋｇ体重の式（Ｉ）の化合物を投与するための、第１項～第２４項のいずれかに記載の医薬組成物。
［２６］約１０～約３００ｍｇ／ｋｇ体重の式（Ｉ）の化合物を投与するための、第１項～第２５項のいずれかに記載の医薬組成物。
［２７］約２５～約２００ｍｇ／ｋｇ体重の式（Ｉ）の化合物を投与するための、第１項～第２６項のいずれかに記載の医薬組成物。
［２８］血栓溶解剤と併用される、第１項～第２７項のいずれかに記載の医薬組成物。
［２９］血栓溶解剤がｔＰＡである、第２８項に記載の医薬組成物。
［３０］血栓溶解剤がアルテプラーゼである、第２８項または第２９項に記載の医薬組成物。
［３１］静脈内投与される、第１項～第３０項のいずれかに記載の医薬組成物。 For example, the following embodiments are provided.
[1] Equation (I):

[During the ceremony,
Ra is halo, hydroxy, alkyl, halo-substituted alkyl, aryl, halo or alkyl-substituted aryl, alkoxy, hydroxy or carboxy-substituted alkoxy, aryloxy, halo or alkyl-substituted aryloxy, CHO, C (O) -alkyl, C (O). ) -Aryl, C (O) -alkyl-carboxyl, C (O) -alkylene-carboxyester and selected from the group consisting of cyano
m is an integer selected from 0 to 4]
A pharmaceutical composition for the treatment of cerebral infarction, which comprises a compound of the above or an ester, oxide, pharmaceutically acceptable salt or solvate thereof.
[2] The pharmaceutical composition according to item 1, wherein Ra is independently selected from the group consisting of halo, hydroxy, alkyl, halo-substituted alkyl and alkoxy.
[3] The pharmaceutical composition according to item 1 or 2, wherein Ra is independently selected from the group consisting of halo and alkyl, respectively.
[4] The pharmaceutical composition according to any one of items 1 to 3, wherein two Ras are present, one is halo and the other is alkyl.
[5] The pharmaceutical composition according to any one of items 1 to 4, wherein the compound of the formula (I) is selected from the compounds shown in Table 1.
[6] The compound of the formula (I) is 4-amino-3- [6- (4-fluoro-2-methylphenyl) pyridin-3-ylazo] naphthalene-1-sulfonic acid, items 1 to 2. The pharmaceutical composition according to any one of paragraph 5.
[7] Any of the items 1 to 6, which comprises 4-amino-3- [6- (4-fluoro-2-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid sodium salt. The pharmaceutical composition described.
[8] The pharmaceutical composition according to any one of paragraphs 1 to 7, wherein the cerebral infarction is lacunar infarction, atherothrombotic cerebral infarction or cardiogenic cerebral infarction.
[9] The pharmaceutical composition according to any one of items 1 to 8, wherein the cerebral infarction is lacunar infarction.
[10] The pharmaceutical composition according to any one of items 1 to 9, which is administered in the acute phase of cerebral infarction.
[11] The pharmaceutical composition according to any one of items 1 to 10, which is administered immediately before or immediately after the reperfusion treatment after cerebral infarction.
[12] The pharmaceutical composition according to any one of items 1 to 10, which is administered at the same time as reperfusion therapy after cerebral infarction.
[13] The pharmaceutical composition according to paragraph 11 or 12, wherein the reperfusion treatment is administration of a thrombolytic agent.
[14] The pharmaceutical composition according to paragraph 11 or 12, wherein the reperfusion procedure is surgery.
[15] The pharmaceutical composition according to any one of paragraphs 1 to 14, which is administered once.
[16] The pharmaceutical composition according to any one of items 1 to 14, which is administered a plurality of times.
[17] The pharmaceutical composition according to any one of paragraphs 1 to 14, which is continuously administered.
[18] The pharmaceutical composition according to any one of items 1 to 17, which is administered within about 24 hours from the onset of cerebral infarction.
[19] The pharmaceutical composition according to any one of items 1 to 18, which is administered within about 12 hours from the onset of cerebral infarction.
[20] The pharmaceutical composition according to any one of items 1 to 19, which is administered within about 8 hours from the onset of cerebral infarction.
[21] The pharmaceutical composition according to any one of items 1 to 20, which is administered within about 6 hours from the onset of cerebral infarction.
[22] The pharmaceutical composition according to any one of paragraphs 1 to 21, which is administered within about 4.5 hours from the onset of cerebral infarction.
[23] The pharmaceutical composition according to any one of items 1 to 22, which is administered within about 3 hours from the onset of cerebral infarction.
[24] The pharmaceutical composition according to any one of items 1 to 23, for administering the compound of the formula (I) having a body weight of about 1 to about 1000 mg / kg.
[25] The pharmaceutical composition according to any one of items 1 to 24, for administering the compound of the formula (I) having a body weight of about 5 to about 500 mg / kg.
[26] The pharmaceutical composition according to any one of items 1 to 25, for administering the compound of the formula (I) having a body weight of about 10 to about 300 mg / kg.
[27] The pharmaceutical composition according to any one of paragraphs 1 to 26, for administering the compound of formula (I) having a body weight of about 25 to about 200 mg / kg.
[28] The pharmaceutical composition according to any one of items 1 to 27, which is used in combination with a thrombolytic agent.
[29] The pharmaceutical composition according to claim 28, wherein the thrombolytic agent is tPA.
[30] The pharmaceutical composition according to claim 28 or 29, wherein the thrombolytic agent is alteplase.
[31] The pharmaceutical composition according to any one of items 1 to 30, which is administered intravenously.

All references cited in this disclosure are part of this disclosure by explicit source.
All of the above description is non-limiting and can be modified without departing from the scope of the invention as defined in the appended claims. In addition, the examples below are all non-limiting examples and are provided solely to illustrate the invention.

Materials and Methods Primary Neuron Cultures Cerebral neuron cultures were prepared from 17-day-old Sprague dolly rat embryos (Shimizu laboratory supplies) using previously described methods (Maki T, et al, Annals of Clinical and). Translational Neurology, 2014 Aug; 1 (8): 519-33). This will be explained briefly. The cortex was removed and separated. Cells 200,000-250,000 on a dish coated with poly-D-lysine in Dulbecco's Modified Eagle's Medium (DMEM) containing 5% heat-immobilized fetal bovine serum and 1% penicillin / streptomycin. The seeds were sown at a density of ² pieces / cm2. Twenty-four hours after seeding, the medium was replaced with Neurobasal (NB) medium containing 0.5 mM glutamine, 1% penicillin / streptomycin and 2% B27 supplement. Cultured neurons were used in the experiment 14 days after sowing.

The oxygen-glucose-deficient medium was replaced with glucose-free DMEM. The cells were then placed in a sealed Anaero container (Mitsubishi Gas Chemical Company) equipped with an Anaero Pack for 1.5-2 hours. After oxygen-glucose deficiency, cells were transferred to normal medium and returned to a 5% CO ₂ incubator with normal oxygen pressure.

Cell viability test Cell viability was evaluated by the Cell Counting Kit-8 (CCK-8) assay (Dojin Chemical Laboratory). The CCK-8 assay is a water-soluble tetrazolium salt 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium. It is based on the fact that sodium salts (WST-8) are converted to water-soluble formazan dyes when reduced by dehydrogenase in the presence of electron carriers. Cells were incubated with 10% CCK-8 solution for 1-2 hours at 37 ° C. Then, the absorbance of the culture medium was measured with a microplate reader (measurement wavelength 450 nm, reference wavelength 630 nm).

Immunocytochemistry Cells were washed twice with phosphate buffered saline (PBS) followed by treatment with 4% paraformaldehyde (PFA) for 15 minutes. After washing twice with PBS, the cells were incubated with PBS / 0.1% Tween (10 minutes) and blocked with 3% BSA / PBS (1 hour at room temperature). Cells were incubated overnight with a primary antibody against MAP2 (1: 1000, Sigma Aldrich, M1406) at 4 ° C. Subsequently, after washing with PBS, the cells were incubated with the secondary antibody for 1 hour at room temperature. Finally, the nuclei were counterstained with DAPI.

ATP Assay Using a cellular ATP test reagent (Toyo Ink Mfg. Co., Ltd.) based on luciferase chemiluminescence, ATP levels in cell lysates were measured according to the manufacturer's protocol. This will be explained briefly. 100 μL of lysate provided by the manufacturer was added to the cells in each well of a 96-well plate. After incubating for 5 minutes at room temperature, the luminescence of the aliquots of the solution was measured with a luminometer.

Western blot cells were harvested and RIPA buffer (20 mM HEPES-KOH pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet-) containing 10% 2-mercaptoethanol (Nakalitesk) and 1% protease inhibitor (Nakalitesk). Dissolved in P40, 1% sodium deoxycholate). The excised brain sample was homogenized in RIPA buffer containing 2-mercaptomethanol and a protease inhibitor. The sample was sonicated and centrifuged at 4 ° C. and 15,000 rpm for 15 minutes and the supernatant was collected. Protein (15-20 μg / lane) was loaded, separated by 5-20% SDS-PAGE and transferred to polyvinylidene chloride membrane (Millipore). The primary antibodies are: β-actin (1: 5000, Sigma Aldrich, A5441), CHOP (1: 1000, Cell Signaling Technology, L63F7), NeuN (1: 2000, Merck Millipore, ABN78), VCP. (1: 500, ABGENT, AP6920b). HRP-labeled secondary antibody (Santa Cruz Biotechnology) was used for visualization by enhanced chemiluminescence (Nacalai Tesque).

Adult male C57BL / 6 (C57BL / 6NJcl) and CB-17 (CB-17 / lcr-+ / + Jcl) mice (6-7 weeks old) were purchased from Shimizu Laboratory Supplies and Claire Japan, respectively. Free intake of food and water.

Methods described prior to stroke surgery (Kasahara Y, Ihara M, Nakagomi T, et al. A highly reproducible model of cerebral ischemia / reperfusion with extended survival in CB-17 mice. Neuroscience Research, 2013 Jul; 76 (3): A distal middle cerebral artery occlusion (distal MCAO) was performed in CB-17 mice with a slight modification of 163-8). This will be explained briefly. General anesthesia was induced by inhalation of 4% isoflurane (Pfizer) and maintained by inhalation of 1.5% isoflurane. Mice were placed in the lateral decubitus position and an incision was made in the skin between the left eyeball and the left ear canal. The left salivary gland and part of the temporalis muscle were resected so that the middle cerebral artery (MCA) was visible through the skull. A burr hole was made in the skull. The MCA was then separated and transiently occluded with a monofilament nylon thread. After 22 minutes of occlusion, removal of the nylon thread restored MCA blood flow. Rectal temperature was monitored during surgery and maintained at 36.0-37.2 ° C with a feedback controlled heating pad.

In C57BL / 6 mice, the previously described method (Doyle KP, Fathali N, Siddiqui MR, et al. Distal hypoxic stroke: a new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit. Journal of Neuroscience. Methods, 2012 May 30; 207 (1): 31-40) were modified to perform distal MCAO with hypoxia. Mice were placed in a large chamber containing 10% oxygen and 90% nitrogen after occlusion of the distal MCA with nylon thread. After 30 minutes of hypoxic conditions, the nylon thread was removed and returned to normal oxygen pressure conditions. Pseudosurgery was the same as stroke surgery, except for the obstruction of the distal MCA.

Assessment of Stroke Volume Mice were deeply anesthetized 24 hours after ischemia and transcardially perfused with PBS and 4% PFA. Brain was removed and fixed after 48 hours in 4% PFA. Further cryoprotection in 20% sucrose until the brain sank to the bottom of the vial. For Nistle staining, the brain was continuously cut with a cryostat and sliced on a slide with a thickness of 20 μm every 600 μm (+2.0, +1.4, +0.8, anterior-posterior to cross suture, +0.8, +0.2, -0.4, -1.0 and -1.6 mm). Sections were incubated in Cresyl Violet for 15 minutes and dehydrated in 70% methanol for 5 seconds. The slide was then covered with a mounting medium. The area of the ipsilateral hemisphere and the area of the infarct in each section were measured using NIH imageJ software. The measured value was multiplied by the distance between the sections (600 μm) and summed throughout the brain to obtain a volume measurement.

Accelerated rotarod test Twenty-four hours after ischemia, mice were placed on an accelerated rotarod device (accelerating from 0 rpm to 40 rpm over 4 minutes). The time that the mouse stayed on the rotating cylinder was measured. Tried 3 times and used the maximum latency to fall.

Administration of KUS121 4-amino-3- [6- (4-fluoro-2-methylphenyl) pyridine-3-ylazo] naphthalene-1 produced by the method described in International Publication No. 2012/014994 (Patent Document 1). -Sodium sulfonic acid salt (referred to as KUS121 or KUS) was dissolved in PBS containing 5% Cremophor EL (Sigma) to prepare a 20 μg / μL solution. A mouse model of localized ischemic stroke was administered KUS121 solution intravenously and intraperitoneally (KUS121 100 mg / kg and 50 mg / kg, respectively).

Statistical analysis Unless otherwise noted, all values are expressed as mean ± SE. Statistical analysis was performed using Dunnett's test (cell survival test) and Student's t-test (tests other than cell survival test). Differences of p <0.05 were considered statistically significant differences.

result
KUS121 protects primary cerebral cortical neurons under oxygen-glucose deficient conditions by preventing ATP depletion
The procedure of the in vitro experiment using the primary cerebral cortex neurons is shown in FIG. To evaluate the neuronal protective effect of KUS121 under ischemic conditions, cell survival tests were performed after oxygen glucose deficiency (OGD) treatment in the presence and absence of KUS121 (Fig. 1, top). OGD was performed in rat cerebral cortex primary neuron cultures for 2 hours, followed by recovery in 21% O ₂ and glucose-containing medium for 22 hours. Control neurons were maintained in 21% _O2 , glucose-containing medium for 24 hours. Exposure to OGD caused cell death of primary cerebral cortical neurons. However, the presence of 100 μM and 200 μM KUS121 during the experiment significantly improved the viability of neurons after OGD treatment (18.1 ± 1.9% in medium (DMSO), 43.5 ± in 100 μM KUS). 3.1%, 42.4 ± 3.7% at 200 μM KUS; p <0.001) (Fig. 2). In addition, 100 μM KUS121 significantly increased the MAP2 positive area compared to medium (DMSO) (0.87 ± 0.13% for medium (DMSO), 2.64 ± 0.41% for 100 μM KUS). P <0.05) (Fig. 3). DMSO or KUS121 without OGD treatment did not affect neuronal viability.

Since KUS121 has been reported to be able to prevent ATP depletion in pathological situations, it was investigated whether KUS121 had a similar effect on OGD-treated primary cerebral cortical neurons. After 1.5 hours of OGD treatment, cell ATP levels were measured by a luciferase-based assay. Treatment with KUS increased ATP levels (14.3 ± 1.1% in medium (DMSO), 28.0 ± 2.3% in 100 μM KUS; p <0.001) (FIG. 4). DMSO or KUS121 without OGD treatment had no effect on ATP levels. These data suggest that KUS121 protects primary cerebral cortical neurons under oxygen-glucose deficient conditions by preventing ATP depletion.

KUS121 protects primary cerebral cortical neurons under ER stress-induced conditions Since ER stress is induced after ischemia, the protective effect of KUS121 when the primary cerebral cortical neurons were treated with tunicamycin was tested (Fig. 1, bottom). ). Tunicamycin treatment is known to cause ER stress. C / EBP homologous protein (CHOP) is a central mediator of ER stress-induced cell death and is upregulated under ER stress. Cerebral cortical neurons were exposed to 0.25 μg / mL tunicamycin for 6 hours. KUS121 suppressed the expression of CHOP in primary cerebral cortical neurons treated with tunicamycin (Fig. 5).

The procedure of an in vivo experiment using mice in which treatment with KUS121 improves motor function and reduces cerebral infarct volume is shown in FIG. To evaluate the effect of KUS121 on cerebral ischemia, transient localized cerebral ischemia was induced in C57BL / 6 mice. Immediately after the distal occlusion of the left middle cerebral artery, KUS121 was administered (Fig. 6, top). Twenty-four hours after occlusion, KUS121 significantly prolonged dwell time on the rotarod compared to the medium (134.5 ± 18.4 seconds on medium (5% Cremophor), 201.0 ± 21 on KUS). .8 seconds; p <0.05) (Fig. 7). In addition, infarct volume was significantly reduced by KUS121 treatment (8.8 ± 0.63% for medium (5% Cremophor), 4.7 ± 1.70% for KUS; p <0.05) (FIG. 8). ).

The neuroprotective effect was confirmed in CB-17 mice. Administration of KUS121 immediately prior to ischemia (bottom of FIG. 6) reduced infarct volume (11.8 ± 0.60% with medium (5% Cremophor), 5.74 ± 1.64% with KUS; p <0. .05) (Fig. 9). In addition, Western blots of the cerebral cortex revealed that expression of the neuron marker NeuN was conserved by KUS121 (Fig. 10).

The present disclosure provides a method for treating cerebral infarction by a mechanism of action that has not been used so far, and can be used in the medical field.

Claims (11)

Equation (I):

[During the ceremony,
Ra is halo, hydroxy, alkyl, halo-substituted alkyl, aryl, halo or alkyl-substituted aryl, alkoxy, hydroxy or carboxy-substituted alkoxy, aryloxy, halo or alkyl-substituted aryloxy, CHO, C (O) -alkyl, C (O). ) -Aryl, C (O) -alkyl-carboxyl, C (O) -alkylene-carboxyester and selected from the group consisting of cyano
m is an integer selected from 0 to 4]
A pharmaceutical composition for the treatment of cerebral infarction, which comprises a compound of the above or an ester, oxide, pharmaceutically acceptable salt or solvate thereof. The pharmaceutical composition according to claim 1, wherein Ra is independently selected from the group consisting of halo, hydroxy, alkyl, halo-substituted alkyl and alkoxy. The compound of formula (I)
4-Amino-3- (6-phenylpyridine-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- (6-p-toluylpyridine-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- (6-m-toluylpyridine-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- (6-o-toluylpyridine-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- (6-biphenyl-2-ylpyridine-3-ylazo) naphthalene-1-sulfonic acid;
3- [6- (2-Acetylphenyl) Pyridine-3-ylazo] -4-aminonaphthalene-1-sulfonic acid;
3- [6- (3-Acetylphenyl) Pyridine-3-ylazo] -4-aminonaphthalene-1-sulfonic acid;
3- [6- (4-Acetylphenyl) Pyridine-3-ylazo] -4-aminonaphthalenesulfonic acid;
4-Amino-3- [6- (2,4-dichlorophenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-trifluoromethylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-trifluoromethylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-chlorophenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-chlorophenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-chlorophenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-methoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-Methoxyphenyl) Pyridine-3-ylazo] Naphthalene-1-sulfonic Acid;
4-Amino-3- [6- (2-isopropoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-isopropoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-phenoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-methoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2,3-dimethylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2,5-dimethylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3,5-dimethylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-trifluoromethylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4- {4- [5- (1-Amino-4-sulfonaphthalene-2-ylazo) pyridin-2-yl] phenyl} -4-oxobutylic acid;
4-Amino-3- (6-biphenyl-3-ylpyridine-3-ylazo) naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-cyanophenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-cyanophenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3,5-bistrifluoromethylphenyl) pyridine-3-ylazo] naphthalene sulfonic acid;
4-Amino-3- [6- (4-benzoylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-propoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-fluoro-2-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (5-fluoro-2-propoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-fluoro-6-propoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-fluoro-2-propoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (5-fluoro-2-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-fluoro-5-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-butoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-hexyloxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-Butylphenyl) Pyridine-3-ylazo] Naphthalene-1-sulfonic Acid;
4-Amino-3- [6- (2-hydroxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3-{6- [2- (6-hydroxyhexyloxy) phenyl] Pyridine-3-ylazo} Naphthalene-1-sulfonic acid;
4- {2- [5- (1-Amino-4-sulfonaphthalene-2-ylazo) pyridin-2-yl] phenoxy} butyric acid;
4-Amino-3-{6- [2- (3-hydroxypropoxy) phenyl] Pyridine-3-ylazo} Naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2-isobutoxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (5-chloro-2-hydroxyphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4-methylbiphenyl-2-yl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4'-chloro-4-methylbiphenyl-2-yl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (4,3', 5'-trimethylbiphenyl-2-yl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3'-chloro-4-methylbiphenyl-2-yl) pyridin-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (2,6-dimethylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
4-Amino-3- [6- (3-formyl-2-isopropoxy-5-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid; and
4-Amino-3- [6- (3-formyl-2-butoxy-5-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid;
The pharmaceutical composition according to claim 1 or 2, which is selected from the group consisting of. Any of claims 1 to 3, wherein the compound of formula (I) is 4-amino-3- [6- (4-fluoro-2-methylphenyl) pyridine-3-ylazo] naphthalene-1-sulfonic acid. The pharmaceutical composition according to. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cerebral infarction is lacunar infarction, atherothrombotic cerebral infarction or cardiogenic cerebral infarction. The pharmaceutical composition according to any one of claims 1 to 5, which is administered at the time of reperfusion after cerebral infarction. The pharmaceutical composition according to any one of claims 1 to 6, which is administered within about 8 hours from the onset of cerebral infarction. The pharmaceutical composition according to any one of claims 1 to 7, which is administered within about 4.5 hours from the onset of cerebral infarction. The pharmaceutical composition according to any one of claims 1 to 8, which is used in combination with a thrombolytic agent. The pharmaceutical composition according to claim 9, wherein the thrombolytic agent is tPA. The pharmaceutical composition according to any one of claims 1 to 10, which is administered intravenously.

Images